Zwitterionic Polypeptide-protein Conjugation for the Safe and Efficient Delivery of Therapeutic Enzymes PROJECT SUMMARY One of the major obstacles that impede the wide application of therapeutic protein products is their potential immunological response, especially for those obtained from non-human sources. Currently, the most successful strategy to mitigate immune response induced by foreign proteins is ?PEGylation?, i.e., to shield the protein surface epitopes with polyethylene glycol (PEG). This surface conjugation strategy has been shown to decrease to some extent immune responses to the underlying protein and more than ten PEGylated protein products have been approved by the Food and Drug Administration (FDA). However, recent studies both in animal models and clinical trials have demonstrated the presence of induced anti-PEG antibodies after repeated administrations and pre-existing anti-PEG antibodies, which directly challenge the future of this PEGylation technology. We believe there are two shortcomings for the current PEGylation technology: 1) the haptenic character of amphiphilic PEG leading to anti-PEG antibodies and 2) non-degradable character of amphiphilic PEG leading to vacuolation of kidney and liver. Funded by one NIH R21 grant before, we successfully developed a zwitterionic poly(carboxybetaine) (PCB) protected uricase, which was shown to be capable of maintaining protein bioactivity, reducing the immunogenicity of encased proteins and extending their circulation time without induced anti-polymer Abs. In the course of our study, we discovered zwitterionic poly(EK) (PEK), a biodegradable polypeptide comprising of alternative lysine (K) and glutamic acid (E). Extended from the zwitterionic concept, `highly water binding ability and zero net charge', zwitterionic PEK has all properties of zwitterionic PCB. But, PEK is degradable to promote its elimination from the body while PEG or PCB is not. A biodegradable zwitterionic polymer is highly desirable. Thus, PEK is a great alternative beyond PEGylation, but has never been studied for its application to drug delivery. The purpose of this R21 proposal is to explore the potential of degradable zwitterionic poly(EK) (PEK)-protein conjugates. As a degradable version of PCB, PEK is anticipated to provide similar benefits as PCB to its modified protein drugs while possessing excellent biodegradability. In this proposal, uricase, a non-human enzyme with strong immunogenicity, will be used as the protein drug. We hypothesize and will confirm though this study that PEK-uricase conjugates will provide a safe and efficient strategy for enzyme delivery by greatly reducing immunogenicity, improving pharmacokinetics (PK), and enhancing pharmacodynamics (PD). Since PEGylation has been widely used in protein therapeutics, successful completion of this project will culminate in a new approach as an alternative to or a replacement over the PEGylation technology, producing safer and more effective enzyme therapeutics.